Synthesis and Functionalization of Poly(ethylene oxide-b-ethyloxazoline) Diblock Copolymers with Phosphonate Ions

Alfred Yuen-Wei Chen

Thesis submitted to the Faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Master of Science In

Macromolecular Science and Engineering

Judy S. Riffle, Chair

James E. McGrath

Richey M. Davis

September 6, 2013

Blacksburg, Virginia

Keywords: poly(ethylene oxide), poly(2-ethyl-2-oxazoline), ion encapsulation, phosphonate, phosphonic acid, nanoparticles, functionalized random copolymer

Synthesis and Functionalization of Poly(ethylene oxide-b-ethyloxazoline) Diblock Copolymers with Phosphonate Ions

Alfred Yuen-Wei Chen

Abstract

Poly(ethylene oxide) (PEO) and poly(2-ethyl-2-oxazoline) (PEOX) are biocompatible

polymers that act as hydrophilic “stealth” drug carriers. As block copolymers, the PEOX group

offers a wider variety of functionalization. The goal of this project was to synthesize a

poly(ethylene oxide)-b-poly(2-ethyl-2-oxazoline) (PEO-b-PEOX) block copolymer and

functionalize pendent groups of PEOX with phosphonic acid. This was achieved through

cationic ring opening polymerization (CROP) of 2-ethyl-2-oxazoline monomer onto PEO. These

polymerizations used tosylsulfonyl chloride as initiator. Size-exclusion chromatography (SEC)

was used to determine the molecular weights of the block copolymers. Two samples of 1:2 and

one sample of 1:3 of PEO-to-PEOX block copolymers were made. These samples underwent

partial hydrolysis of the PEOX pendent groups to form the random block copolymer,

poly(ethylene oxide)-b-poly(2-ethyl-2-oxazoline)-co-poly(ethyleneimine) (PEO-b-PEOX-co-

PEI). These reactions showed that there was a degree of control based on the moles of acid.

Diethyl vinyl phosphonate was attached to the nitrogen of PEI units via Michael addition where

the phosphorylation left <1% of PEI units unattached. The ethyl groups on the phosphonates

were further hydrolyzed off phosphonate with HCl acid leaving phosphonic acid. After each step

of synthesis, structures and composition were confirmed using 1H NMR. Due to the nature of the

phosphonic acid, the polymer can be utilized in the incorporation and release of cationic drugs.

iii

Acknowledgements

I’d like to express my earnest and sincere gratitude to Dr. Judy Riffle for her guidance,

patience, and generous support during my time here at Virginia Tech even before I attended as a

graduate student. I would not have known how wonderful a place Virginia Tech, Blacksburg,

and Virginia was without her letting me become part of her research group and growing as a

student and as an individual. It has been a great honor and a fulfilling experience to be working

for her. I’d also like to thank Dr. Richey Davis and Dr. James McGrath for their insightful

perspectives and knowledge as committee members and as professors in my coursework.

I would also like to give thanks to Oguzhan Celebi for his wisdom and intuitive input

during these years of research and friendship outside the lab. I especially would like to thank

Oguzhan for his efforts and support in my experiments and significantly streamlining my

reaction efforts and writing critiques. I would like to thank my friends and colleagues in Dr.

Riffle’s research group: Suzanne Barnes, Nan Hu, Jue Liang, Dr. Yinnian Lin, Nipon Pothayee,

and Rui Zhang. Thank you and I wish good luck for you all.

I’d also like to give thanks to Philip Huffstetler, Tim Vadala, Mike Heinser, Kevin Myer,

Kevin Ortman and Nikorn Pothayee. You all made my experience in Virginia Tech some of the

most rewarding I’ve had. Thank you for inspiring me to come to Virginia Tech and see all the

people and opportunities. I’d especially like to thank Philip Huffstetler as his insight and energy

was what inspired me to be a part of Dr. Riffle’s research group. I’m grateful for the love and

support that I’ve received from my parents, Dr. Albert and Grace Chen.

iv

Table of Contents

CHAPTER 1: Introduction....................................................................................................... 1 CHAPTER 2: Literature Review ............................................................................................. 2

2.1 Overview ...........................................................................................................................2

2.2 Poly(2-oxazoline)s .............................................................................................................2

2.2.1 Introduction .................................................................................................................2

2.2.2 Structure, Synthesis and Properties ..............................................................................3

2.2.3 Applications in Biomedicine........................................................................................6

2.2.4 Stimuli Responsive Polyoxazolines .............................................................................8

2.2.5 Polyoxazoline in Lipopolymers ................................................................................. 10

2.2.6 Polyoxazoline Vesicles .............................................................................................. 12

2.2.7 Gene Transfer Chemistry ........................................................................................... 12

2.2.8 Antimicrobial Polyoxazoline ..................................................................................... 14

2.3 Phosphonates ................................................................................................................... 15

2.3.1 Introduction ............................................................................................................... 15

2.3.2 Chemistry of Phosphonate and Phosphonic acid ........................................................ 16

2.3.3 Biomedical Applications of Phosphonate ................................................................... 18

2.3.4 Phosphonates as Biocompatible Surface Media ......................................................... 20

CHAPTER 3: Synthesis and Functionalization of Poly(ethylene oxide-b-2-ethyl-2-oxazoline) Diblock Copolymers with Phosphonate Ions ............................................................................. 24

3.1 Synopsis .......................................................................................................................... 24

3.2 Experimental ................................................................................................................... 25

3.2.1 Materials ................................................................................................................... 25

3.2.2 Synthesis of Tosylated PEO Macroinitiator ............................................................... 25

3.2.3 Synthesis of Poly(ethylene oxide-b-2-ethyl-2-oxazoline) ........................................... 26

3.2.4 Acid Hydrolysis of Poly(ethylene oxide-b-2-ethyl-2-oxazoline) ................................ 26

3.2.5 Synthesis of Phosphonic Acid Functionalized PEO-b-PEOX-co-PEI ......................... 27

3.2.6 Characterization ........................................................................................................ 28

3.3 Results and Discussion .................................................................................................... 29

3.3.1 Synthesis of Poly(ethylene oxide-b-2-ethyl-2-oxazoline) and Modifications to Form Copolymers Containing Ammonium Phosphonate Zwitterions ........................................... 29

v

3.3.2 Copolymer Compositions Before and After Hydrolysis and Post-phosphorylation ..... 33

3.3.3 Influence of Modification on Thermal Stability ......................................................... 41

CHAPTER 4: Conclusions and Future Works ...................................................................... 43 REFERENCES........................................................................................................................ 44

vi

List of Figures

Figure 2.1 Arrangements of cyclic imino ether ...........................................................................3 Figure 2.2 Monomer Structures, Tg, and Surface Energies of poly(2-n-alkyl 2-oxazoline) vs. Carbon Number. Image reproduced from Poly(2-oxazoline)s: Alive and Kicking [2] Used under Fair Use, 2013 .............................................................................................................................5 Figure 2.3 Polymerization of 2-ethyl-2-oxazoline with a tosylate initiator and termination with potassium hydroxide ...................................................................................................................7 Figure 2.4 Representation synthesis of poly(2-oxazoline)/DSPE conjugates ............................. 11 Figure 2.5 Structures of antimicrobial macromers..................................................................... 15 Figure 2.6 Basic structure of phosphonate compound ............................................................... 16 Figure 2.7 Reaction catalyzed by PEP mutase .......................................................................... 18 Figure 2.8 Examples of commercially used pesticides .............................................................. 19 Figure 2.9 Structures of phosphorus–based chemical weaponry based on methylphosphonates . 19 Figure 2.10 Diagram of “Tethering by Aggregation and Growth” (T-BAG) using Phosphonic Acid Solutions to Coat Titanium Oxide surfaces ....................................................................... 22 Figure 3.1 Synthesis of poly(ethylene oxide-b-2-ethyl-2-oxazoline) copolymers ...................... 29 Figure 3.2 Synthesis of diethyl phosphonate derivatives of poly(ethylene oxide)-poly(2-ethyl-2-oxazoline) diblock copolymers through acid hydrolysis and Michael addition ........................... 30 Figure 3.3 Conversion of diethyl phosphonate group to phosphonic acid .................................. 31 Figure 3.4 SEC Viscosity measurement of PEO-b-PEOX in comparison to PEO ...................... 32 Figure 3.5 1H NMR PEO 5K and PEO-b-PEOX diblock in HDO ............................................. 34 Figure 3.6 1H NMR Comparison illustrating pH influence on polymer mixture ........................ 36 Figure 3.7 1H NMR of PEO-b-(PEOX-co-PEI) phosphorylation ............................................... 38 Figure 3.8 1H NMR of diethyl phosphonate conversion to phosphonic acid .............................. 39 Figure 3.9 TGA graph depicting diblock series from PEO-b-PEOX to phosphonic acid modified final form .................................................................................................................................. 41

vii

List of Tables

Table 2.1 pKa Values of unsubstituted alkylphosphonic acids in water ..................................... 17 Table 2.2 pKa Values of aliphatic bisphosphonic acids in water ............................................... 17 Table 3.1 Compositions of PEO to PEOX in copolymers .......................................................... 35 Table 3.2 PEO-b-PEOX hydrolysis to form PEO-b-PEOX-co-PEI ........................................... 35 Table 3.3 Compositions of PEO-b-PEOX-co-PEI-co-phosphonate ........................................... 40

1

CHAPTER 1: Introduction

In the field of drug delivery, it is imperative that nanoparticle design can adapt to various

biological media and incorporate different facets of drugs such as anionic or cationic properties.

These biocompatible polymeric carriers are often composed of amphiphilic block copolymers

due to their ubiquitous nature and often controllable properties during and after synthesis.

Poly(ethylene oxide) (PEO) is a biocompatible polymer component known for its hydrophilicity,

low cytotoxicity, and low immunogenicity. By incorporating PEO in nanoparticle designs, size

can be increased allowing for drug-carrying nanoparticles to avoid filtration via the kidneys

while keeping consistent biodistribution even while circulating the blood stream. Poly(2-ethyl-2-

oxazoline) (PEOX) is also another biocompatible polymer that exhibits similar “stealth”

behaviors but can also be functionalized with biocompatible pendent substituents. In this thesis,

phosphonic acid was used in replacing the pendent ethyl groups of PEOX. The anionic quality of

the phosphonic acid has the potential to be pH controllable and provide an environment where

cationic drugs and contrast agents can be held.

The second chapter reviews the controlled radical ring-opening polymerization and

medicinal benefits of polyoxazolines and the biomedical implications of phosphonates. The

Third chapter will describe the synthesis and characterization of PEO-b-PEOX block copolymers

and the steps taken to functionalize the copolymers. This includes pendent group hydrolysis,

Michael addition, and ethyl hydrolysis of phosphonate as well as 1H NMR characterizations.

2

CHAPTER 2: Literature Review

2.1 Overview

The topics discussed in this literature review are separated into two sections. The first

will describe the synthesis, properties and applications of polyoxazolines and its diverse usage in

biomedicine. The second section will discuss phosphonates and the properties and chemistry are

beneficial in biological systems.

2.2 Poly(2-oxazoline)s

2.2.1 Introduction

In recent years, the demand for more versatile biocompatible nanotechnology has

directed attention to the growing research focused on the design of “smart” polymers. Before the

1990’s, polyoxazolines had relatively limited uses in industry, mostly in coating and paint

dispersants [1]. These materials can be prepared by cationic polymerization in solution with

conventional thermal heating or under microwave conditions [2-4]. Today, polyoxazolines have

been shown to have a wide variety of uses, especially in the biomedical field due to their low

cytotoxicity, tunable solution properties, resilience to degradation and uptake in the body, and

the ability to be incorporated in a multitude of nanostructures [1, 2, 5, 6].

When developing materials for drug delivery, there are a variety of problems that stem

from the complexity of biological systems. Some of these involve administration methodology,

properties of the drug and how it will interact in biological media, target sites, and desired drug

release rates [7, 8]. Even afterwards, drug designs must take into account inter- and

intramolecular interactions that can cause harm to the biological system if left unchecked such as

3

waste removal functions, immunogenicity and organ accumulation [8, 9]. As a result, the designs

of vehicles for drug delivery are complex while conversely, and ironically, such pharmaceutics

need to be as simple as possible to avoid unnecessary side reactions. Biocompatible polymers

such as poly(ethylene oxide) and poly(2-oxazoline)s are ideal since they provide versatile

synthetic polymer platforms whereby complex structures can be attained while allowing for

incorporating more complex groups [5, 9].

2.2.2 Structure, Synthesis and Properties

The monomer structure of polyoxazoline is the oxazoline ring which is comprised of

nitrogen, oxygen, and a double bonded carbon that is arranged as a 5-membered ring. This cyclic

imino ether comes in three distinct arrangements that differ from one another by the carbon-

carbon bond, though 2-oxazoline has been the most widely used monomer in polymerizations (2)

(Figure 2.1) [10].

ON

ON ON

R

R R1 2 3

R = alkyl or phenyl group

Figure 2.1 Arrangements of cyclic imino ether

While substituents can be present in the 4- and 5- positions, experiments, conducted by

Saegusa and Kobayashi showed that this impeded polymerizations through steric crowding, often

requiring specific catalysts or spatial arrangements to perform successful polymerizations [1].

4

Polyoxazolines are polymerized by cationic ring opening polymerization (CROP). First

developed in 1966 by four independent groups, polyoxazoline synthesis follows a living cationic

ring opening polymerization [9, 11-14]. It is initiated when the nitrogen of the ring, a weak

nucleophile, substitutes onto the initiator thereby releasing a weak base. This leaves the positive

charge distributed over the nitrogen, carbon and oxygen of the ring. Propagation occurs by

subsequent repeated attack on the carbon in the 5-position [4, 13]. Termination is induced after

the polymerization by adding another nucleophile such as hydroxide ion or an amine.

Well-defined polyoxazoline copolymers can be synthesized with narrow molecular

weight distributions [15]. A crucial factor determining the polyoxazoline properties is the length

of the 2-substituted chain. Hoogenboom et al. has reported experiments with libraries of various

polyoxazoline monomers differentiated by the chain length of the 2- group that ranged from 1 to

9 carbons [2]. Three key points can be drawn from their results: (1) Side-chain length does not

significantly affect the rate of polymerization of homopolymers except for when the substituent

is methyl. Methyloxazoline polymerizes somewhat faster due to a higher nucleophilicity. (2)

Dynamic Scanning Calorimetry (DSC) analysis showed that poly(2-alkyl-2-oxazoline)s that had

side chains longer than butyl are semicrystalline, whereas polymers with ethyl and butyl side

chains were completely amorphous. The glass transition temperatures (Tg) of polymers with

pentyl and longer chains were not identified and this was attributed to the small changes in heat

capacities at the glass transitions. (3) Surface energies, calculated by measuring contact angles of

diiodomethane and ethylene glycol as test liquids, were influenced by chain length. Polymers

with methyl to propyl chains had high surface energies of around 45 mN/m and those with longer

chain lengths had low surface energies of around 22 mN/m [2].

5

Figure 2.2 Monomer Structures, Tg, and Surface Energies of poly(2-n-alkyl 2-oxazoline) vs. Carbon Number. Image reproduced from Poly(2-oxazoline)s: Alive and Kicking [2] Used under

Fair Use, 2013

Figure 2.2 details the surface energy of the polymers with regards to the length of the

side chain and their glass transition temperatures. A sharp transition in surface energy correlates

with a shift from the propyl to pentyl groups along the ambient temperature line (20 OC)

suggesting that chain mobility may influence the capability for the side chain to populate the

surface [16].

Another class of polyoxazolines is random copolymers from monomers with different

side chain lengths. Park and Kataoka showed that different polyoxazoline compositions can

influence properties [17]. For example, the melting point of poly(nonyloxazoline) at 150 oC

6

decreases linearly upon incorporation of ethyloxazoline. This indicates disturbance of the

crystalline phase and results in the absence of crystallinity when more than 20 wt% of

ethyloxazoline is incorporated into the chain. Likewise, Tg’s for poly(methyloxazoline) and

poly(ethyloxazoline) decrease with incorporation of nonyloxazoline. Surface energies for the

copolymers were ~42 mN/m until the copolymers were comprised of ~70% wt% of

nonyloxazoline. Interestingly, with high nonyloxazoline content, the Tg was near ambient

temperature. At this stage, the surface energy decreased as the Tg dropped below ambient

temperature due to the surface coverage by the nonyl side chains [2].

2.2.3 Applications in Biomedicine

In spite of their structural similarities there are certain core features that make

polyoxazolines in general attractive for biomedicine. The potential for polymers to be effective

vehicles for drugs has been a major driving force in the development of novel polyoxazolines.

Synthetic polymers are attractive for drug and gene delivery due to the capability to tailor

structure and properties. Polymers can improve the efficacy and bioavailability of drugs by

protecting them from degradation and immune response, and improving solubility. Containment

in the polymer vehicles can also be used to tailor and sustain drug release rates. Polyoxazolines

have been incorporated and assembled into nanoscale structures such as micelles, liposomes, and

hydrogels. It has also been shown that these polymers can avoid rapid immune response, similar

to that observed polyethylene oxide [18].

Saccharide initiators have been used to polymerize 2-oxazolines [19]. This was achieved

by first preparing a saccharide-substituted oxazoline ring, then forming a cationic oxazolinium

initiator through the reaction of the saccharide-substituted ring with methyl triflate. The

7

saccharide-oxazoline cation was then reacted with a variety of 2-oxazoline monomers to produce

polymers with a saccharide end group at the initiator end.

Figure 2.3 Polymerization of 2-ethyl-2-oxazoline with a tosylate initiator and termination with

potassium hydroxide

Poly(2-methyl-2-oxazoline)s were demonstrated to be biocompatible by Goddard et al [6,

20]. A series of poly(2-methyl-2-oxazoline)-polyethylene glycol copolymers labeled with 125I

8

were injected intraveneuosly into mice. It was observed that after 24 hours, 7% of 15 kDa

copolymers remained in the blood while 28% of 29 kDa copolymers remained after the same

time period. Gaertner et al., discovered that poly(methyloxazoline) and poly(ethyloxazoline)

with 111In labels had negligible accumulation in any organs, except for the kidneys, but were

quickly excreted [21]. Intravenously injected doses had only 0.8% (polymethyloxazoline) and

1% (polyethyloxazoline) left in the blood after 3 hours. Total accumulation activity per kidney

was 0.4% and 0.6% for polymethyloxazoline and polyethyloxazoline, respectively, suggesting

that excretion of the polymers is mostly done through the kidneys. In spite of this, investigations

were conducted comparing with the clearance rates with two different nuclear tracers of kidney

functions, Tc-diethylenetriaminepentaacetate (DTPA) and Tc-mercaptoacetyltriglycine (MAG3).

It was shown that DPTA is filtered exclusively through renal glomerular filtration making the

rate equal to glomerular filtration. MAG3 excretion is affected by glomerular filtration and tubuli

secretion making it equivalent to the renal plasma flow rate. When comparing to the 5 kDa

polyoxazolines, the clearance rates were much slower than the nuclear tracers suggesting that

there were specific interactions between the polymers and proteins in the plasma. Overall,

polyethyloxazoline was retained longer than polymethyloxazoline, and this was attributed to its

more hydrophobic nature.

2.2.4 Stimuli Responsive Polyoxazolines

Stimuli responsive polymers ideally change properties drastically in response to a small

change in environmental conditions. Most biological stimuli consist of changes in pH and/or salt

and biochemical concentrations. It is well documented that polyoxazoline copolymers can

respond in this manner [22-24]. The self-assembling nature of amphiphilic poly(ethyloxazoline)

9

copolymers is controlled by their structure and chemical environment. An example of this is with

a series of poly(2-ethyl-2-oxazoline)-poly(ε-caprolactone) alternating multiblock copolymers

prepared from short preformed blocks (approximately 1000-2500 g/mol). These act as hydrogels

even though there are no covalent crosslinks. The copolymers were hydrophilic but water

insoluble and exhibited reversible swelling behavior upon heating and cooling. Swelling ratios

were dependent on the temperature, volume fractions of the blocks, and the length of the

polyethyloxazoline block. At low temperatures (~15 °C), hydration of the hydrophilic

polyethyloxazoline primarily dictated the swelling ratio. As temperature was increased

(approaching 35 °C), maintaining the hydrophobic interactions from polycaprolactone within the

gel became more important [1, 25]. Polyethyloxazoline has been shown to exhibit LCST

behavior in water, but the short blocks in this study had LCST transitions outside of the range

investigated.

Poly-2-ethyl-2-oxazoline-b-ε-caprolactone diblock copolymers have also been

investigated. These copolymers form micelles in water with critical micelle concentrations of 1.0

to 8.1 mg/L. The outer shell is comprised of the hydrophilic poly(2-ethyl-2-oxazoline) block, and

the shell is known to form complexes with polymethacrylic acid through hydrogen bonds. The

carbonyl oxygen and nitrogen on the polyoxazoline are attracted to the carboxylic acids on the

polymethacrylic acid due to hydrogen bonding [15, 26]. When pH drops below 3.5, the micelles

precipitate, and upon increase of pH to 3.8 they can be redispersed.

The thermoresponsive nature of polyoxazolines has been investigated [9, 23, 27-29].

They become insoluble in water as temperature is raised through the lower critical solution

temperature (LCST) and cloud points are observed. At low temperatures, the polymer dissolves

in water due to hydrogen bonds. As temperature is increased, the hydrogen bonds are weakened

10

causing the polymers to aggregate due to the entropy gain associated with water dissociation

[28]. The alkyl chain length of the polyoxazoline influences the LCST. Kwei et al. studied

polyethyloxazoline thermosensitvity where cloud points were found to depend on polymer

concentration and molecular weight (20-500 kDa). These factors correlated with the lower

critical solution temperature (LCST) range of 61 - 64 °C. It was also found that addition of salts

to these solutions influenced the LCST. Sodium chloride addition was found to decrease the

LCST and cause precipitation, but conversely, LCST was increased with addition of

tetrabutylammonium bromide. However, it must be noted that these tests were performed with a

very high molecular weight sample of 500k [30].

Hoogenboom et al. measured the LCST transition of polyoxazolines with varying

hydrophilicity, with and without the presence of added Hofmeister salts. As expected, the LCST

increased with increasing hydrophilicity in the absence of the salts. These salts included NaSCN,

NaClO4, LiClO4, NaI, LiI, NaCl, NaOAc, LiOAc, Na2SO4, and Li2SO4. It was demonstrated that

addition of -SCN, ClO4-, and I- increased cloud points. By contrast, Cl-, -OAc, and SO4

2- resulted

in lower cloud points. The anions were found to have a greater effect than the cations. The cloud

points of the most hydrophilic polymer studied, polyethyloxazoline, could be varied over a very

broad range in temperature by adding these salts [31].

2.2.5 Polyoxazoline in Lipopolymers

Micelles and liposomes are in high demand due in part to the convenience of being able

to self-assemble in water around a desired drug through the use of amphiphilic copolymers. The

most commonly studied vehicles have involved poly(ethylene glycol) (PEG). There have been a

number of cases where PEG-antibody immune responses have occurred [32]. Thus, Woodle et

11

al., prepared poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline) lipo-polyoxazoline

conjugates in combination with distearoylphosphatidyl ethanolamine (DSPE) as shown in Figure

2.4 [33].

Figure 2.4 Representation synthesis of poly(2-oxazoline)/DSPE conjugates

These lipophilic polyoxazolines with 67Ga labels were injected into the bloodstream of

rats. They showed equivalent retention in the bloodstream as well as exhibiting steric colloidal

stabilization due to high chain mobility and hydrophilicity. The hydrophilic oxazoline

components of these materials suppressed their interaction with compounds in the blood that

would cause immune response.

12

2.2.6 Polyoxazoline Vesicles

Polyoxazoline-based amphiphilic block copolymers are particularly attractive due to their

self-assembly into micelles and vesicles. These nanocontainers have been used for a number of

different applications. One such application is the encapsulation of calcium ions [34]. Poly(2-

methyl-2-oxazoline)-b-polydimethylsiloxane-b-poly(2-methyl-2-oxazoline) (PMOXA-PDMS-

PMOXA) was prepared and bound to platinum and glass electrodes in phosphate buffer [1]. This

resulted in phosphate ions becoming incorporated into the micelle or vesicle. These membranes

containing phosphate ions were incubated with calcium chloride in the presence of ionophores,

and this lead to precipitation of calcium phosphates in the vesicle [1].

2.2.7 Gene Transfer Chemistry

Polyoxazolines can be converted to polyethyleneimine through hydrolysis of the pendent

amides. The polyethyleneimine exists as a polycation at low pH. These polycations have been

investigated as components of complexes with anionic polynucleic acids. Gene delivery has been

investigated for over 25 years using both viruses and polycations to carry the DNA. [1, 35, 36].

An example of this is with a poly(ethylene glycol)-b-poly(ethylene imine). Synthesis of the PEG

used a heterotelechelic poly(ethylene oxide) with an acetal group on one end and a mesylate on

the other. This polymer was used as a macroinitiator for the cationic ring-opening polymerisation

of 2-methyl-2-oxazoline to nominally make an acetal-PEG-PMOXA polymer. This was

hydrolyzed using strong aqueous base to give the reported acetal-PEG-PEI. 1H NMR confirmed

the expected composition of the polyethyleneoxide-polyoxazoline block copolymer. However, it

is not clear in light of the work described in this thesis that efficient initiation of the

macroinitiator to form the block copolymer would have occurred under the conditions specified.

13

The NMR showed that the hydrolysis reaction to form the polyethyleneimine block was

quantitative. Park et al. described the synthesis of poly(2-ethyl-2-oxazoline)-co-

poly(ethyleneimine) random copolymers through partial acid hydrolysis of poly(2-ethyl-2-

oxazoline) [37]. Increasing the acid concentration afforded polymers with increasing degrees of

hydrolysis and therefore increasing charge densities on the backbone. These materials formed

tightly compacted complexes with DNA. A 50k g/mol polyethyloxazoline that had been 88.0%

hydrolyzed was found to have the most efficient complexation capacity. However, the high

degree of hydrolysis also increased cytotoxicity. The higher the charge and molecular weight, the

more cytotoxic the polymer became [38].

PEI has also been shown to have potential as a component of carriers for anticancer

agents [39]. Folate receptors are proteins that are overexpressed on tumor cells. A folate-

poly(ethyleneimine)-b-poly(L-lactide) copolymer was synthesized by preforming a partially

hydrolyzed polyethyloxazoline oligomer terminated with amine groups. This was reacted with a

carboxyfunctional poly-L-lactide oligomer, then further reacted with folate. A polymer/DNA

complex was prepared with the folate-poly(ethyleneimine)-b-poly(L-lactide). It was found that

the folate-poly(ethyleneimine)-b-poly(L-lactide) reduced toxicity relative to PEI but maintained

acceptable transfection efficiency.

Triblock copolymers that integrated PEG to improve colloidal stability and decrease

cytotoxicity have been reported. However, these authors did not provide any evidence that the

molecular weights could be controlled. Nevertheless, the polymethyloxazoline componenet was

hydrolyzed to yield PEI blocks, and the resultant materials were complexed with DNA. When

screened for transfection efficiency, these copolymers resulted in improved transfection

efficiency over PEI with less cytotoxicity [40].

14

2.2.8 Antimicrobial Polyoxazoline

Antimicrobial polymers may have advantages relative to traditional low-molecular-

weight antimicrobial agents due to lower toxicity and less development of microbial resistance

[41]. In a Minimum Inhibitory Concentration study done by Waschinski and Tiller,

polyethyloxazoline and polymethyloxazoline were end-functionalized with various alkyl groups

at the initiator end and various quaternary ammonium salts at their terminal end, and these were

investigated as antimicrobial agents. Only the polymethyloxazolines exhibited antimicrobial

activity. It was found that the potency of the materials depended on the end-group structures at

both ends and also depended on the aggregate characteristics. The materials that were below

their critical micelle concentrations (CMC) were more active than those above the CMC. It was

hypothesized that the both of the endgroups interacted with the cell membranes, thereby

destabilizing the membranes [1, 42, 43].

Tiller et al. have investigated polymethyloxazoline as an additive for a contact-active

acrylate-based antimicrobial material without the release of low molecular weight antimicrobial

agents into the environment [42, 44]. The polymer was initiated with a biocidal benzyl bromide

functional initiator, and terminated with a acrylamide functional tertiary amine. These

macromers were copolymerized with hydroxyethylmethacrylate and 1,3-glyceroldimethacrylate.

The polymethyloxazoline chain was utilized as a spacer for the biocidal copolymers. Figure 2.5

shows the structure of the polymer macromonomers used. The study demonstrated that by

weight, the copolymer containing as little as 0.4% of the 2400 g/mol Mn macromer was highly

antimicrobial [44].

15

Figure 2.5 Structures of antimicrobial macromers

2.3 Phosphonates

2.3.1 Introduction

Two industrial and biologically significant classes of organic compounds are

phosphonates and phosphonic acids. They are known to have a wide variety of applications

especially in medicine, finishes for paper and textiles, and in agriculture. However, the exact

biological role of phosphonates is not yet fully understood. The first documented experiment

with phosphonates was in 1865 with the synthesis of bisphosphonates used in textiles and

fertilizers [45]. Naturally occurring phosphonates were discovered in rumen protozoa in 1959

leading to further investigations of their presence in fungi, bacteria, and higher order organisms

such as the snail schistosome vector, Biomphalaria [46, 47]. Furthermore, because of the affinity

that phosphonates have with metal ions, extensive research has been focused on bone

regeneration utilizing chelating properties. As a result, phosphonates are used as osteoporosis

drugs [48]. The biocompatible nature also has provided a bridging aspect between inorganic and

organic media. One long-term impact of the research described in this thesis may be to utilize the

16

unique properties of polymers containing phosphonates to complex with cations. These may be

applicable for bioimaging and drug delivery.

2.3.2 Chemistry of Phosphonate and Phosphonic acid

The structure of phosphonate is a phosphorus atom covalently bonded to two hydroxide

or ester variants, double bonded to oxygen, and with a bond to an alkyl or aryl group as shown in

Figure 2.6.

POR1

O

R2O

R3

R1, R2, R3 = H, Alkyl, or Aryl Groups Figure 2.6 Basic structure of phosphonate compound

The oxygen substituents make the molecule soluble in water and provide a negative

electronic environment to function as chelating agents. Phosphonates are often soluble in both

water and organic solvents, while phosphonic acids are typically water-soluble but poorly

soluble in organic solvents. Organophosphonates have a highly stable carbon-phosphorus bond

strong enough to resist hydrolysis, thermal decomposition, and photolytic degradation. This is

due to high activation energies despite similarities of the bond energies between a C-P and C-C

bond which were determined to be 64 kcal and 62 kcal, respectively.

Phosphonic acid is a moderately strong acid and dibasic. Table 2.1 presents the pKa

values of alkylphosphonic acids and Table 2.2 shows the pKa values of aliphatic bisphosphonic

acids. According to Table 2.1, the pKa values increase with growing numbers of carbon atoms

17

and branching degree. The pKa’s of aliphatic bisphosphonic acids, shown in Table 2.2, are

affected by the length of the connecting carbon chains [49].

Table 2.1 pKa Values of Unsubstituted Alkylphosphonic Acids in Water

Alkyl Group pK1 pK2 pK2 - pK1 Methyl 2.33 7.76 5.43

2.35 7.1 4.75 2.38 7.74 5.36 2.48 7.34 4.86

Ethyl 2.39 7.98 5.59 2.45 7.85 5.4 2.43 8.05 5.62

n-Propyl 2.45 8.06 5.61 2.49 8.18 5.69

Isopropyl 2.55 7.75 5.2 2.66 8.44 5.78

n-Butyl 2.59 8.19 5.6 Isobutyl 2.7 8.43 5.73

sec-Butyl 2.74 8.48 5.74 t-Butyl 2.79 8.88 6.09

Neopentyl 2.84 8.65 5.81 1,1-Dimethylpropyl 2.88 8.98 6.08

n-Hexyl 2.6 7.9 5.3 2.4 8.25 5.85

n-Dodecyl * 8.25 --- 1.22† 0.81† 5.59

* Low solubility of this compound in acid solutions prevented the determination of pKa † Values represented are on the molal scale and were obtained in 50 % wt ethanol

Table 2.2 pKa Values of Aliphatic Bisphosphonic Acids in Water

Compound pK1 pK2 pK3 pK4 H2O3P(CH2)4PO3H2 <2 2.75 7.54 8.38 H2O3P(CH2)3PO3H2 <2 2.65 7.34 8.35

H2O3PCH2CH(CH3)PO3H2 <2 2.6 7.00 9.27 H2O3PCH2PO3H2 <2 2.57 6.87 10.33

18

2.3.3 Biomedical Applications of Phosphonate

The vast majority of natural and synthetic products involving phosphorus are phosphate

esters. Phosphonate derivatives are also significant in industry and biologically relevant products

[50]. Horiguchi and Kandatsu in 1959 were able to isolate 2-aminoethylphosphonate in rumen

protozoa [51]. It was later discovered that the phosphonate was an integral component of the

phosphonolipid structure in several varieties of sea organisms and in the human brain [50, 52].

Further studies by Seidel and Bowman discovered the enzyme phosphoenolpyruvate

phosphonomutase (PEP mutase) which converts phosphates into phosphonates (Figure 2.7) [47,

53].

Figure 2.7 Reaction catalyzed by PEP mutase

The stereochemistry of the oxygen atoms allow for effective chelation of many harmful

heavy metal cations in ground water such as lead, copper, and arsenic [54-56]. This affinity has

been exploited as an oxidation stabilizer which has benefited the paper, textiles, and wood

varnishing industries. The C-P bond coupled with strong cationic attraction of oxygen atoms has

resulted in oligomeric phosphonates that, when coated onto wood or plastic casings, are resilient

to combustion [57].

Phosphonates have also influenced the development of pesticides. Pesticide brand names

such as Round UpTM (glyphosate) utilize an aminoalkyl phosphonate as part of the active group.

Pesticide development has been integral to phosphonate research. Butonate, trichlorfon, and o,o-

19

bisphenyl methylphosphonate are a few examples of phosphonate insectides and fungicides [58].

(Figure 2.8)

Figure 2.8 Examples of commercially used pesticides

The toxic nature of such pesticides, however, garnered interest from the military as

biochemical weaponry. Gerhard Schrader introduced the deadly potential in 1936 of phosphorus-

containing agents in the form of the first nerve agent, Tabun. Various dialkyl

methylphosphonates were precursors to various nerve agents including Sarin, and Soman [59].

(Figure 2.9)

Figure 2.9 Structures of phosphorus–based chemical weaponry based on methylphosphonates

20

The field of medicine has greatly benefited from the functional potential of phosphonates.

Therapeutic agents derived from acyclic nucleoside phosphonates were shown to be effective

against certain invasive pathogenic species and microorganisms [60-62]. Several examples

include Tenofovir disoproxil fumarate, an HIV treatment agent that is also effective against

hepatitis B, Adefovir, a nucleotide reverse transcriptase inhibitor used against the Herpes virus,

and Cidofovir, which treats the Human papilloma virus. Specifically, HIV and hepatitis C

reproduction were inhibited through phosphonate interactions with proteases [59, 63, 64].

Alafosfalin is an antibiotic known to inhibit cell wall biosynthesis and it is potentially useful in

the gastroenteritis treatment and bacterial urinary tract infections [59, 65, 66].

One subset of phosphonates that has been particularly useful in the clinic is

bisphosphonates where two phosphonate groups are attached to a single carbon. These are used

as treatments for bone related disease including osteoporosis and even bone and breast cancers

[67-70]. In addition, bisphosphonates have shown promise in their ability to chelate

radionucleotides and iron oxide contrast agents in an effort to gain further insight into cancers

and their related biological processes [71, 72].

2.3.4 Phosphonates as Biocompatible Surface Media

Many implantable materials and devices need to be interfaced with biological

components. This is particularly significant with metallic prosthetics. Exposure of the implants to

the body can lead to adsorption of proteins, cell-surface interactions, or tissue development or

regrowth. This makes the physical and biochemical properties of the implant surface a high

priority for design. Phosphonic acids and phosphonates can produce a biocompatible interphase

region to bridge metal and tissue. Titanium metal, used in orthopedics and dental implants, has

21

excellent mechanical properties. Titanium oxide is an inert compound that is easily integrated in

bone regeneration [73, 74]. This is a key reason why it is used in dentistry. However, complete

incorporation with the bone lattice via chemical bonding has not been observed. Titanium oxide

and siloxane surfaces can be functionalized with phosphonates to produce self-assembled

monolayers [73, 75]. These surfaces can then be further functionalized with cell-binding peptides

to allow for successful surface interactions with bone tissue [76]. Phosphonic acid has only

recently been utilized to form bioactive titanium surfaces and these show promise as they are

biocompatible, are more hydrolytically stable than silane surfaces, and can be bound to various

material surfaces [75, 76].

Gawalt et al. first reported the self-assembly of alkanephosphonates onto titanium oxide

surfaces. In general, the surface of titanium oxidizes easily forming an oxide surface that is

resistant to surface chemical modifications [73]. However, after aerosol application and solvent

removal of alkanephosphonate in THF, heating the surface to 120 °C for 18 hours formed an

alkanephosphonate film. This film was shown to be highly resistant to solvent removal and

mechanical peeling.

However, investigations of cell-adhesion proteins interacting with phosphonic acid-

modified titanium surfaces showed a substantial increase in immune responses. It was

hypothesized that phosphonic acids were indiscriminately binding to many surface proteins of

cells causing distortion and irritation at the interface [77]. Research conducted by Adden et al.

sought to better integrate bone tissue with various phosphonic acid compounds in order to

prevent aseptic loosening. This is an immune response where tiny particles between the joint and

tissue cause inflammatory responses that can lead to bone degradation and loosening of the

implant. (11-Hydroxyundecyl) phosphonic acid and (12-carboxydodecyl) phosphonic acid were

22

deposited onto silicon or titanium substrates by submersing these substrates in solution at a

concentration below the critical micelle concentration. As the solvent evaporated, a phosphonate

film became firmly attached on the substrate due to the partially organized aggregation at the

surface. Figure 2.10 presents a diagram of the procedure.

OH

PO

OO

P OO O

CHO O

Phosphonic AcidSolution

Sample Holders

Titanium OxideSurface Phosphonic Acid

Film

Slow Solvent Evaporation

TiO2 TiO2

Ti TiTi

TiO2

(11-Hydroxyundecyl)

PhosphonicAcid

1. 1.

2. Heat2. Heat

(12-carboxydodecyl)

PhosphonicAcid

Figure 2.10 Diagram of “Tethering by Aggregation and Growth” (T-BAG) using Phosphonic Acid Solutions to Coat Titanium Oxide surfaces

A similar procedure has been used to immobilize cell-adhesive RGD peptides and BMP-2

proteins onto phosphonate-modified titanium oxide surfaces. BMP-2 protein enhances bone

growth by differentiating mesenchymal cells into osteoblasts. Two methods were devised [76].

23

First, (11-hydroxyundecyl) phosphonic acid was immobilized then activated for protein binding

with carbonyldiimidazole. Second, (12-carboxydodecyl) phosphonic acid was bound in a similar

manner except with the use of N-hydroxysuccinimide to activate binding at the surface. Human

BMP-2 proteins were bound to the carboxy group through their primary amine, forming a

biocompatible cell adhesion layer that was specific for the osteoclast cells and promoted bone

growth. Phosphonic acid was shown to be chemically and physically more resilient to

degradation and aseptic loosening than the previously used silanes.

24

CHAPTER 3: Synthesis and Functionalization of Poly(ethylene

oxide-b-2-ethyl-2-oxazoline) Diblock Copolymers with Phosphonate

Ions

3.1 Synopsis

This chapter discusses the synthesis, functionalization, and characterization of PEO-b-

PEOX as it is converted into the phosphonic acid copolymer form. In recent years, amphiphilic

block copolymers have been extensively investigated as potential drug carriers due to their self-

assembly into various kinds of morphological structures in aqueous solutions. However, PEOX

blocks can be additionally functionalized with other biocompatible molecules such as proteins.

The phosphonic acid substituents provide an electronegative environment for cationic drugs,

proteins, and metal ions to be held in. Such complexes can induce micelle or vesicle formation

depending on the structure of the block ionomer, changes in pH, temperature, and ionic strength.

In this study, we demonstrate a novel approach to prepare poly(ammonium phosphonate) block

copolymers with PEO. This builds upon previous work that describes converting poly(2-

oxazolines) to linear PEI or poly(2-oxazoline-co-PEI) [37, 78, 79]. PEO-b-PEOX block

copolymers were synthesized using cationic ring-opening polymerization with a tosylate

initiator. Of the three samples, two weight ratios were made: 1:2 and 1:3 PEO to PEOX. SEC,

however, revealed slow initiation relative to propagation though further functionalizations were

not affected by this. The samples were hydrolyzed with HCl to form the random copolymer

PEO-b-PEOX-co-PEI. These copolymers were dialyzed in pH ~9 to free the nitrogen of the PEI

group which would allow for Michael addition onto diethyl vinyl phosphonate forming PEO-b-

PEOX-co-PEI-co-phosphonate copolymers. Further acid hydrolysis of the phosphonate forms the

25

final product, PEO-b-PEOX-co-PEI-co-phosphonic acid. Due to the ionic nature of the

phosphonic acid these novel materials may bind strongly to opposite charged molecules and

metal ions.

3.2 Experimental

3.2.1 Materials

Poly(ethylene oxide) methyl ether (Mn = 5800 g/mol), p-toluenesulfonyl chloride (TsCl,

>98%), anhydrous methanol (>99.8%), calcium hydride (>90%), and manganese (II) chloride

tetrahydrate (99.9%) were purchased from Aldrich and used as received. 2-Ethyl-2-oxazoline

and chlorobenzene, both from Aldrich, were dried over CaH2. 2-Ethyl-2-oxazoline was vacuum

distilled into a dry flask under nitrogen. Chlorobenzene was distilled under nitrogen.

Triethylamine (TEA, 99.5%) was obtained from Fluka. Bromotrimethylsilane (TMS-Bromide,

97%) and aqueous potassium hydroxide (1 M) were purchased from Alfa Aesar and used as

received. Dialysis tubing (3500 g/mol MWCO) was obtained from Spectra/Por. Aqueous

hydrochloric acid (2 M, LabChem) was used as received. Diethyl vinyl phosphonate (98%) was

obtained from Epsilon Chiminie. Diethyl ether, methanol and dichloromethane (Fisher

Scientific) were used as received. Anhydrous dichloromethane (>99.8%) was obtained from

EMD Chemicals.

3.2.2 Synthesis of Tosylated PEO Macroinitiator

The synthesis of PEO methyl ether tosylate macroinitiator is provided. A 5800 g/mol Mn

PEO oligomer (30.2 g, 5.21 mmol) was added to a 250-mL flask equipped with a magnetic stir

bar and enclosed with a rubber septum bound with steel wire. The PEO oligomer was vacuum

dried at 70°C for 24 h and dissolved in dichloromethane (70 mL). Et3N (3.67 mL, 26.3 mmol)

was added to serve as an acid scavenger. In a separate flask, TsCl (2.04 g, 10.7 mmol) was

26

dissolved in 50 mL of CH2Cl2. The tosyl chloride solution was syringed into the reaction flask.

The reaction was stirred for 24 h at 25 °C. The reaction mixture was passed through a 0.2-μm

Teflon® filter, then transferred to a separatory funnel, and washed with DI water 4X to remove

salts. The organic phase was concentrated via rotary evaporation, and the resulting macroinitiator

was collected via precipitation into diethyl ether, and then was dried overnight under vacuum at

50 °C.

3.2.3 Synthesis of Poly(ethylene oxide-b-2-ethyl-2-oxazoline)

Double hydrophilic poly(ethylene oxide-b-2-ethyl-2-oxazoline) (PEO-b-PEOX) block

copolymers were synthesized via cationic polymerization of 2-ethyl-2-oxazoline from the end of

the tosylated PEO macroinitiator. An representative procedure for preparing a targeted

composition of ~2.5:1 wt:wt PEO:PEOX is as follows. A tosylated PEO macroinitiator (3.3 g,

0.57 mmol) was dried overnight at 80 °C in a 100-mL flask equipped with a magnetic stir bar

and enclosed with a rubber septum. Chlorobenzene (20 mL) was added to dissolve the PEO

macroinitiator, and then 2-ethyl-2-oxazoline (5.51 mL, 5.41 g, 55 mmol) was syringed into the

macroinitiator solution. The reaction was performed at 110 °C for 24 h. The reaction mixture

was cooled to room temperature and the polymer chains were terminated with 1 M KOH (1 mL,

1 mmol) in methanol. The diblock copolymer was isolated by precipitation into diethyl ether, and

collected by filtration and dried at 50 °C in vacuo overnight.

3.2.4 Acid Hydrolysis of Poly(ethylene oxide-b-2-ethyl-2-oxazoline)

An acid hydrolysis procedure for removing a portion of the pendent amide groups from

the PEOX blocks is provided. A PEO-b -PEOX diblock copolymer with 5800 g/mol PEO:14,300

g/ mol PEOX (5 g, 0.25 mmol, 36 meq of amides) was charged to a 100-mL flask containing a

27

magnetic stir bar and enclosed with a septum. HCl(aq) (2 M, 10.5 mL, 21 mmol) and 4.5 mL of

DI water were syringed into the flask. The reaction was conducted at 90 °C and maintained for

24 h, then cooled to room temperature. The reaction mixture was diluted to 50 mL with DI water

and placed in a 3500 g/mol MWCO cellulose acetate dialysis membrane and dialyzed against 4 L

of DI water for 48 h. The pH of the receptor medium was adjusted to ~ 9 with KOH to remove

acetates and other salts. The contents of the dialysis membrane were transferred to a 250-mL

flask and freeze-dried. 1H NMR was used to confirm the extent of hydrolysis.

3.2.5 Synthesis of Phosphonic Acid Functionalized PEO-b-PEOX-co-PEI

The ethyleneimine groups of the partially hydrolyzed copolymers were reacted via

Michael addition with diethyl vinyl phosphonate. A representative procedure for addition of

phosphonate groups to the PEO-b-PEOX-co-PEI copolymer is as follows. A PEO-b-PEOX-co-

PEI (4.07 g, 0.25 mmol, 19.3 meq of amides, 16.7 meq of imines) was charged to a 100-mL flask

equipped with a stir bar, and dissolved in DI water (10 mL). Diethyl vinyl phosphonate (7.75

mL, 8.22 g, 50 mmol) was added to the polymer solution. The reaction was performed at 80 °C

for 24 h. The reaction mixture was diluted with DI water (40 mL) and placed in a 3500 g/mol

MWCO cellulose acetate dialysis bag and dialyzed against 4 L of DI water for 48 h to remove

excess diethyl vinyl phosphonate. The contents of the dialysis membrane were transferred to a

250-mL flask and freeze-dried.

PEO-b-PEOX-co-PEI-phosphonic acid was prepared by removal of the ethyl ester groups

from PEO-b-PEOX-co-PEI-phosphonate [80, 81]. In a representative procedure, PEO-b-PEOX-

co-PEI-phosphonate (0.50 g, 0.031 mmol, 0.625 meq of phosphonate groups, 1.25 meq of ethyl

groups) was dissolved in anhydrous dichloromethane (5 mL) in a flame-dried 100-mL flask

equipped with a stir bar, and enclosed with a rubber septum. TMS-Br (0.25 mL, 0.29 g, 1.88

28

mmol) was syringed into the reaction flask and stirred at 25 °C for 24 h, then the solvent and

excess TMS-Br were removed via rotary evaporation. The product was further processed under

vacuum at 60 °C for 4 h utilizing a KOH trap system. The resultant polymer was dissolved and

reacted in methanol (3 mL) for 6 h to cleave the trimethylsilyl groups. PEO-b-PEOX-co-PEI-

phosphonic acid was recovered by precipitation into diethyl ether and vacuum-dried at 25 °C for

24 h.

3.2.6 Characterization

1H NMR spectral analyses of polymers were performed using a Varian Inova 400 NMR

spectrometer operating at 400 MHz. The NMR parameters included a pulse width of 30° and a

relaxation delay of 1 s at room temperature with 32 scans. All spectra of the polymers were

obtained in D2O at a concentration of 0.05 g/mL.

Size exclusion chromatography was performed using an Agilent Technologies 1260

Infinity series HPLC pump equipped with a degasser, autosampler, and temperature controlled

column compartment. The detectors were a Dawn Heleos-II multi-angle laser light scattering

detector and Optilab T-rEX refractive index detector both by Wyatt Technologies. The mobile

phase was N-methylpyrrolidone containing 0.05 M LiBr and the stationary phase consisted of

two Alpha M mixed bed columns from Tosoh Bioscience. The column compartment was

maintained at 80 °C and the detectors at 50 °C. Samples were dissolved at approximately 1

mg/mL and filtered with a 0.2 µm Teflon® filter prior to sample loading.

The thermal decomposition behavior of polymer samples was determined using a

thermogravimetric analyzer (TGA, TA Instruments, TGA Q5000) with a heating rate of 10°C/min

under nitrogen. TGA measurements were conducted from 50 to 600°C. Prior to each

29

measurement, all samples were held at 100 °C for 15 min in the TGA instrument to remove any

moisture.

3.3 Results and Discussion

3.3.1 Synthesis of Poly(ethylene oxide-b-2-ethyl-2-oxazoline) and Modifications to

Form Copolymers Containing Ammonium Phosphonate Zwitterions

Syntheses of the PEO-b-PEOX diblock copolymers were performed using cationic ring-

opening polymerization of 2-ethyl-2-oxazoline from one end of PEO-tosylate macroinitiators as

depicted in Figure 3.1.

Figure 3.1 Synthesis of poly(ethylene oxide-b-2-ethyl-2-oxazoline) copolymers

30

After polymerization was complete, the cationic chain ends were neutralized with KOH

in methanol at 25 °C to form a hydroxyl endgroup. A portion of the pendent amides were then

hydrolyzed to obtain a series of PEO-b-(PEOX-co-PEI) copolymers (Figure 3.2). It has been

reported that the degree of deacylation on PEOX could be controlled by the stoichiometric molar

ratio of HCl to amide up to a degree of deacylation of 0.4 [78]. Thus, various concentrations of

HCl were used to control the extent of hydrolysis so that copolymers with different relative

amounts of amide versus ethyleneimine units could be investigated. Ethyleneimine units on

PEO-b-(PEOX-co-PEI) were post-functionalized with diethylvinylphosphonate (DEVP) via

Michael addition in water to introduce pendent ammonium phosphonate groups as precursors for

the corresponding phosphonic acids as shown in Figure 3.2.

Figure 3.2 Synthesis of diethyl phosphonate derivatives of poly(ethylene oxide)-poly(2-ethyl-2-oxazoline) diblock copolymers through acid hydrolysis and Michael addition

31

Water likely accelerated the reactions based on literature reports [82]. The reactions were

conducted with 3 equivalents of diethylvinylphosphonate per equivalent of ethyleneimine and

resulted in 92-96% addition. The ethyl esters on the phosphonate pendent groups were removed

by reacting the polymers with an excess of TMS-Br in anhydrous dichloromethane to form

trimethylsilyl-phosphonates (Figure 3.3), then the silylated intermediates were converted to the

corresponding phosphonic acid derivatives by methanolysis. These reactions were conducted

under strict anhydrous conditions to avoid TMS-Br hydrolysis.

Figure 3.3 Conversion of diethyl phosphonate group to phosphonic acid

32

Size Exclusion Chromatography (SEC) was used to analyze the molecular weight

distributions of the copolymers in NMP containing 0.05 M LiBr with multiple detectors

(differential refractive index, viscosity, and multi-angle laser light scattering). The viscosity

curves of these PEO-b-PEOX copolymers were bimodal as represented in Figure 3.4.

Figure 3.4 SEC Viscosity measurement of PEO-b-PEOX in comparison to PEO

The low molecular weight peak coincided with the SEC curve of the PEO-macroinitiator,

and this signified that there was a substantial amount of PEO-macroinitiator remaining. The

refractive index curves had a negligible peak in the macroinitiator region since the refractive

index of the solvent and PEO were very similar. This allowed clear identification of residual

macroinitiator as the source for the low molecular weight peak. It was reasoned that the rate of

33

initiation relative to propagation was too slow to yield well-defined block copolymers. Thus the

materials were blends containing a major amount of the desired diblock copolymer combined

with the residual PEO macroinitiator.

3.3.2 Copolymer Compositions Before and After Hydrolysis and Post-

phosphorylation

1H NMR spectra of the PEO-b-PEOX diblock copolymer-PEO blends provided the PEO

to PEOX compositions as reported in Table 3.1. A representative spectrum is shown in Figure

3.5. Formation of the PEOX end blocks resulted in the appearance of broad peaks in the region

around 3.3-3.5 ppm assigned to the methylene backbone protons of PEOX and pendent group

protons at 2.2 ppm (-CH2-) and 0.9 ppm (-CH3). The experimental compositions determined via

1H NMR were in good agreement with the targeted compositions.

34

Figure 3.5 1H NMR PEO 5K and PEO-b-PEOX diblock in HDO

35

Table 3.1 Compositions of PEO to PEOX in copolymers

PEO-b-PEOX Copolymer

Targeted Composition

(wt:wt)

Experimental PEO to PEOX Composition wt:wt

1 0.37:0.63 0.39:0.61 2 0.37:0.63 0.37:0.63 3 0.28:0.72 0.29:0.71

As the concentration of HCl in the amide hydrolysis modification was increased, the

extent of hydrolysis also increased as listed in Table 3.2, but the exact degrees of hydrolysis that

were achieved were always somewhat smaller than targeted. As discussed by Kem, this may be

related to a polyelectrolyte effect where hydrolysis becomes slower as polymer units become

protonated, especially at higher degrees of hydrolysis [78]. This does, however, provide a means

to prepare copolymers with different degrees of hydrolysis.

Table 3.2 PEO-b-PEOX hydrolysis to form PEO-b-PEOX-co-PEI

Copolymer* Targeted Hydrolysis Experimental Hydrolysis 2A 60.0% 51.4% 3 58.8% 46.0%

2B 40.0% 27.4% * Copolymer 2 described in Table 1 was hydrolyzed with 2 different concentrations of HCl. The third entry was derived from copolymer 3.

1H NMR spectra reflected the pH sensitive character of the PEO-b-(PEOX-co-PEI)

copolymers as shown in Figure 3.6.

36

Figure 3.6 1H NMR Comparison illustrating pH influence on polymer mixture

The hydrolysis by-product after neutralization with KOH, propionate ion, was ionically

bound to secondary ammonium groups on the ethyleneimine units of the backbone. The alkyl

group proton resonances at 1.1 and 2.5 ppm on the associated propionate ions appeared on the 1H

37

NMR spectra (Figure 3.6) when the polymers were dialyzed against deionized water. The

backbone methylene protons on the secondary ammonium and 2-ethyl-2-oxazoline units were

shifted downfield relative to those on the uncharged polymer. When a hydrolyzed copolymer

solution was dialyzed against high pH (~9) water, resonances from the propionate ions

completely disappeared (since they had been removed by dialysis) and resonances due to

uncharged ethyleneimine and 2-ethyl-2-oxazoline units were shifted upfield. This demonstrated

the complete removal of propionate and neutralization of ethyleneimine units to form free

amines. The hydrolysis degrees of the PEOX component were determined by calculating the

relative areas of proton resonances in the ethylenimine units relative to the pendent group

protons of PEOX [83, 84].

A representative spectrum, as shown in Figure 3.7, denotes the difference between the

bare PEI units with phosphorylated. The appearance of the ethyl peaks at 1.2 and 4.0 shows that

the Michael addition was successful.

38

Figure 3.7 1H NMR of PEO-b-(PEOX-co-PEI) phosphorylation

The 1H NMR spectra of the PEO-b-(PEOX-co-PEI-co-phosphonate) copolymers were

used to analyze the relative amounts of oxyethylene, amide, ethyleneimine, and

diethylphosphonatoethyl pendent groups (Table 3.3). Figure 3.8 (top) shows three different

39

characteristic signals for the diethylphosphonatoethyl groups: (1) methylene protons adjacent to

the phosphorus at 1.9 ppm, and (2) ethyl ester group resonances at 4.0 for -CH2- and (3) 1.2 ppm

for -CH3 groups.

Figure 3.8 1H NMR of diethyl phosphonate conversion to phosphonic acid

The resonances at 2.4-2.8 ppm represent a combination of the methylene adjacent to

nitrogen and the backbone methylenes in the phosphorus-containing units (total of 6 protons per

40

unit), and also the backbone methylenes in residual ethyleneimine units (4 protons per unit). The

relative amounts of the phosphorus-containing units and ethyleneimine were calculated by

subtracting the resonance integral at 1.2 ppm from the total integral at 2.4-2.8 ppm. The

remaining part of the integral at 2.4-2.8 ppm was then representative of the small residual

amount of ethyleneimine units. The peak integral at 0.9 ppm due to the methyl groups on the

amides was utilized to calculate the relative amounts of those units. The resonances due to the

ethyleneoxy and backbone amide units overlap, so the relative number of ethyleneoxy units

could only be estimated by subtracting the appropriate component due to the amides from the

total resonance at ~3.2-3.7 ppm.

Table 3.3 Compositions of PEO-b-PEOX-co-PEI-co-phosphonate

Copolymer Ethyleneoxy Amides Phosphorus-containing units Ethyleneimine

2A 120 42 42 2 2B 129 71 23 2 3 119 64 53 2

The conversion to the corresponding phosphonic acid was demonstrated by the

disappearance of the diethylphosphonatoethyl signals at 1.2 (-CH3) and 4.0 (-CH2) ppm as shown

in Figure 3.8 (bottom). In addition, the methylene group proton resonances on the PEOX

backbone as well as the methylene protons that have similar chemical environments (labeled 6, 7

and 9) showed a significant downfield shift that can be explained by the positive charge on the

ammonium ions formed after phosphonic acid modification. The efficiencies of the Michael

addition were high ranging between 92-96% phosphorylation of ethyleneimine units.

41

3.3.3 Influence of Modification on Thermal Stability

Thermogravimetric analyses (TGA) of PEO homopolymers, PEO-b-PEOX copolymers

and their derivatives were conducted under a nitrogen atmosphere (Figure 3.9).

Figure 3.9 TGA graph depicting diblock series from PEO-b-PEOX to phosphonic acid modified

final form

The thermograms show the weight loss profiles of each polymer as they were converted

from the PEO to the PEO-b-(PEOX-co-PEI-co-phosphonic acid). TGA temperature scans

revealed that both PEO and PEO-b-PEOX and the PEO-b-(PEOX-co-PEI) showed similar

degradation behavior and did not lose a significant amount of weight up to 300-330 °C, and then

they completely decomposed without the formation of char. By contrast, the phosphorus-

42

containing copolymers began weight-loss somewhat earlier (220-260 °C) but formed significant

levels of char (10-17%). It has been reported that at elevated temperatures, dialkyl phosphonates

pyrolyze to monoalkyl derivatives, and then condense to form P-O-P crosslinks that result in

char [85-87]. Consistent with this premise, the amount of char after these tests corresponds to the

weight percentages of the pendent phosphonates in these materials.

43

CHAPTER 4: Conclusions and Future Works

The synthetic strategies that have been developed led to PEO-b-PEOX diblock

copolymers utilizing cationic ring-opening polymerization. However, according to SEC data,

using tosylate initiators result in the copolymer being blended with residual PEO-macroinitiator.

It was surmised that the relatively slow initiation of PEOX relative to propagation is the cause.

However, the post-modification reactions to form ammonium phosphonate zwitterions were

facile and controllable. Based on 1H NMR, it was concluded that novel phosphonic acid-

functionalized random copolymers were successfully synthesized. These new materials with

strongly adhesive phosphonate zwitterions may provide strong complexes with many metals and

metal oxides. Manganese metals and magnetite are some of metal complexes that are currently

the focus of such complexation reaction. The PEO component in the copolymers may also

provide steric dispersion stability in water for micellar complexes with metal oxide nanoparticles

in their cores.

44

REFERENCES

1. Adams, N. and U.S. Schubert, Poly(2-oxazolines) in biological and biomedical application contexts. Advanced Drug Delivery Reviews, 2007. 59(15): p. 1504-1520.

2. Hoogenboom, R., Poly(2-oxazoline)s: Alive and Kicking. Macromolecular Chemistry and Physics, 2007. 208(1): p. 18-25.

3. Guerrero-Sanchez, C., R. Hoogenboom, and U.S. Schubert, Fast and "green" living cationic ring opening polymerization of 2-ethyl-2-oxazoline in ionic liquids under microwave irradiation. Chemical Communications, 2006. 0(36): p. 3797-3799.

4. Makino, A. and S. Kobayashi, Chemistry of 2-oxazolines: A crossing of cationic ring-opening polymerization and enzymatic ring-opening polyaddition. Journal of Polymer Science Part A: Polymer Chemistry, 2010. 48(6): p. 1251-1270.

5. Luxenhofer, R., et al., Poly(2-oxazoline)s as Polymer Therapeutics. Macromolecular Rapid Communications, 2012. 33(19): p. 1613-1631.

6. Hoogenboom, R., Poly(2-oxazoline)s: A Polymer Class with Numerous Potential Applications. Angewandte Chemie International Edition, 2009. 48(43): p. 7978-7994.

7. Malmsten, M., Soft drug delivery systems. Soft Matter, 2006. 2(9): p. 760-769. 8. Viegas, T.X., et al., Polyoxazoline: chemistry, properties, and applications in drug delivery.

Bioconjugate Chemistry, 2011. 22(5): p. 976-986. 9. Hoogenboom, R. and H. Schlaad, Bioinspired Poly(2-oxazoline)s. Polymers, 2011. 3(1): p. 467-

488. 10. Kobayashi, S. and H. Uyama, Polymerization of cyclic imino ethers: From its discovery to the

present state of the art. Journal of Polymer Science Part A: Polymer Chemistry, 2002. 40(2): p. 192-209.

11. Tomalia, D.A. and D.P. Sheetz, Homopolymerization of 2-alkyl- and 2-aryl-2-oxazolines. Journal of Polymer Science Part A-1: Polymer Chemistry, 1966. 4(9): p. 2253-2265.

12. Seeliger, W., et al., Recent Syntheses and Reactions of Cyclic Imidic Esters. Angewandte Chemie International Edition in English, 1966. 5(10): p. 875-888.

13. Kagiya, T., et al., Ring-opening polymerization of 2-substituted 2-oxazolines. Journal of Polymer Science Part B: Polymer Letters, 1966. 4(7): p. 441-445.

14. Bassiri, T.G., A. Levy, and M. Litt, Polymerization of cyclic imino ethers. I. Oxazolines. Journal of Polymer Science Part B: Polymer Letters, 1967. 5(9): p. 871-879.

15. Lee, S.C., et al., Synthesis and Micellar Characterization of Amphiphilic Diblock Copolymers Based on Poly(2-ethyl-2-oxazoline) and Aliphatic Polyesters1. Macromolecules, 1999. 32(6): p. 1847-1852.

16. Wiesbrock, F., et al., Investigation of the Living Cationic Ring-Opening Polymerization of 2-Methyl-, 2-Ethyl-, 2-Nonyl-, and 2-Phenyl-2-oxazoline in a Single-Mode Microwave Reactor†. Macromolecules, 2005. 38(12): p. 5025-5034.

17. Guillerm, B., et al., How to Modulate the Chemical Structure of Polyoxazolines by Appropriate Functionalization. Macromolecular Rapid Communications, 2012. 33(19): p. 1600-1612.

18. Bauer, M., et al., Poly(2-ethyl-2-oxazoline) as Alternative for the Stealth Polymer Poly(ethylene glycol): Comparison of in vitro Cytotoxicity and Hemocompatibility. Macromolecular Bioscience, 2012. 12(7): p. 986-998.

19. Aoi, K., H. Suzuki, and M. Okada, Architectural control of sugar-containing polymers by living polymerization: ring-opening polymerization of 2-oxazolines initiated with carbohydrate derivatives. Macromolecules, 1992. 25(25): p. 7073-7075.

45

20. Goddard, P., et al., Soluble polymeric carriers for drug delivery. Part 2. Preparation and in vivo behaviour of N-acylethylenimine copolymers. Journal of Controlled Release, 1989. 10(1): p. 5-16.

21. Gaertner, F.C., et al., Synthesis, biodistribution and excretion of radiolabeled poly(2-alkyl-2-oxazoline)s. Journal of Controlled Release, 2007. 119(3): p. 291-300.

22. Tanaka, T., Collapse of Gels and the Critical Endpoint. Physical Review Letters, 1978. 40(12): p. 820-823.

23. Schmaljohann, D., Thermo- and pH-responsive polymers in drug delivery. Advanced Drug Delivery Reviews, 2006. 58(15): p. 1655-1670.

24. Lambermont-Thijs, H.M.L., et al., Selective partial hydrolysis of amphiphilic copoly(2-oxazoline)s as basis for temperature and pH responsive micelles. Polymer Chemistry, 2011. 2(2): p. 313-322.

25. Lee, S.C., et al., Synthesis and characterization of amphiphilic poly(2-ethyl-2-oxazoline)/poly(&unknown;-caprolactone) alternating multiblock copolymers. Polymer, 2000. 41(19): p. 7091-7097.

26. Kwon, I.C., Y.H. Bae, and S.W. Kim, Electrically credible polymer gel for controlled release of drugs. Nature, 1991. 354(6351): p. 291-293.

27. Zhang, N., R. Luxenhofer, and R. Jordan, Thermoresponsive Poly(2-Oxazoline) Molecular Brushes by Living Ionic Polymerization: Modulation of the Cloud Point by Random and Block Copolymer Pendant Chains. Macromolecular Chemistry and Physics, 2012. 213(18): p. 1963-1969.

28. Diehl, C. and H. Schlaad, Thermo-Responsive Polyoxazolines with Widely Tuneable LCST. Macromolecular Bioscience, 2009. 9(2): p. 157-161.

29. Hruby, M., et al., Polyoxazoline Thermoresponsive Micelles as Radionuclide Delivery Systems. Macromolecular Bioscience, 2010. 10(8): p. 916-924.

30. Lin, P., et al., Solubility and miscibility of poly(ethyl oxazoline). Journal of Polymer Science Part B: Polymer Physics, 1988. 26(3): p. 603-619.

31. Bloksma, M.M., et al., The Effect of Hofmeister Salts on the LCST Transition of Poly(2-oxazoline)s with Varying Hydrophilicity. Macromolecular Rapid Communications, 2010. 31(8): p. 724-728.

32. Tagami, T., et al., CpG motifs in pDNA-sequences increase anti-PEG IgM production induced by PEG-coated pDNA-lipoplexes. Journal of Controlled Release, 2010. 142(2): p. 160-166.

33. Woodle, M.C., C.M. Engbers, and S. Zalipsky, New Amphipatic Polymer-Lipid Conjugates Forming Long-Circulating Reticuloendothelial System-Evading Liposomes. Bioconjugate Chemistry, 1994. 5(6): p. 493-496.

34. Sauer, M., et al., Ion-carrier controlled precipitation of calcium phosphate in giant ABA triblock copolymer vesicles. Chemical Communications, 2001(23): p. 2452-2453.

35. Putnam, D., Polymers for gene delivery across length scales. Nat Mater, 2006. 5(6): p. 439-451. 36. Wagner, E. and J. Kloeckner, Gene Delivery Using Polymer Therapeutics, in Polymer Therapeutics

I, R. Satchi-Fainaro and R. Duncan, Editors. 2006, Springer Berlin Heidelberg. p. 135-173. 37. Jeong, J.H., et al., DNA transfection using linear poly(ethylenimine) prepared by controlled acid

hydrolysis of poly(2-ethyl-2-oxazoline). Journal of Controlled Release, 2001. 73(2–3): p. 391-399. 38. Abdallah, B., et al., A powerful nonviral vector for in vivo gene transfer into the adult mammalian

brain: polyethylenimine. Human gene therapy, 1996. 7(16): p. 1947-1954. 39. Wang, C.-H. and G.-H. Hsiue, Polymer−DNA Hybrid Nanopar cles Based on

Folate−Polyethylenimine-block-poly(l-lactide). Bioconjugate Chemistry, 2005. 16(2): p. 391-396. 40. Zhong, Z., et al., Low Molecular Weight Linear Polyethylenimine-b-poly(ethylene glycol)-b-

polyethylenimine Triblock Copolymers:  Synthesis, Characterization, and in Vitro Gene Transfer Properties. Biomacromolecules, 2005. 6(6): p. 3440-3448.

41. Siedenbiedel, F. and J.C. Tiller, Antimicrobial Polymers in Solution and on Surfaces: Overview and Functional Principles. Polymers, 2012. 4(1): p. 46-71.

46

42. Waschinski, C.J., et al., Influence of Satellite Groups on Telechelic Antimicrobial Functions of Polyoxazolines. Macromolecular Bioscience, 2005. 5(2): p. 149-156.

43. Waschinski, C.J. and J.C. Tiller, Poly(oxazoline)s with Telechelic Antimicrobial Functions. Biomacromolecules, 2004. 6(1): p. 235-243.

44. Waschinski, C.J., et al., Design of Contact-Active Antimicrobial Acrylate-Based Materials Using Biocidal Macromers. Advanced Materials, 2008. 20(1): p. 104-108.

45. Fleisch, H., The role of bisphosphonates in breast cancer: Development of bisphosphonates. Breast Cancer Res, 2002. 4(1): p. 30 - 34.

46. Whitelaw, F.G., et al., 2-Aminoethylphosphonic acid concentrations in some rumen ciliate protozoa. Applied and Environmental Microbiology, 1983. 46(4): p. 951-953.

47. Seidel, H.M., et al., Phosphonate biosynthesis: isolation of the enzyme responsible for the formation of a carbon-phosphorus bond. Nature, 1988. 335(6189): p. 457-458.

48. Fleisch, H., Bisphosphonates in bone disease : from the laboratory to the patient2000, San Diego [u.a.]: Academic Press.

49. Freedman, L.D. and G.O. Doak, The Preparation And Properties Of Phosphonic Acids. Chemical Reviews, 1957. 57(3): p. 479-523.

50. Ternan, N., et al., Review: Organophosphonates: occurrence, synthesis and biodegradation by microorganisms. World Journal of Microbiology and Biotechnology, 1998. 14(5): p. 635-647.

51. Horiguchi, M. and M. Kandatstu, Isolation of 2-Aminoethane Phosphonic Acid from Rumen Protozoa. Nature, 1959. 184(4690): p. 901-902.

52. Alhadeff, J.A. and G.D. Daves, Occurrence of 2-aminoethylphosphonic acid in human brain. Biochemistry, 1970. 9(25): p. 4866-4869.

53. Bowman, E., et al., Catalysis and thermodynamics of the phosphoenolpyruvate/phosphonopyruvate rearrangement. Entry into the phosphonate class of naturally occurring organophosphorus compounds. Journal of the American Chemical Society, 1988. 110(16): p. 5575-5576.

54. Ballinas, M.d.L., et al., Arsenic(V) Removal with Polymer Inclusion Membranes from Sulfuric Acid Media Using DBBP as Carrier. Environmental Science & Technology, 2003. 38(3): p. 886-891.

55. Nowack, B., Environmental chemistry of phosphonates. Water Research, 2003. 37(11): p. 2533-2546.

56. Al Hamouz, O.C.S. and S.A. Ali, Removal of heavy metal ions using a novel cross-linked polyzwitterionic phosphonate. Separation and Purification Technology, 2012. 98(0): p. 94-101.

57. Ellis, W. and R. Rowell, Flame-Retardant Treatment Of Wood With A Diisocyanate and An Oligomer Phosphonate. Wood and Fiber Science, 1989. 21(4): p. 367-375.

58. Guest, D. and B. Grant, THE COMPLEX ACTION OF PHOSPHONATES AS ANTIFUNGAL AGENTS. Biological Reviews, 1991. 66(2): p. 159-187.

59. Barney, R.J., Synthesis and biological evaluation of novel phosphonates, in Chemistry2010, University of Iowa: University of Iowa. p. 241.

60. Shoshani, I., et al., Inhibition of adenylyl cyclase by acyclic nucleoside phosphonate antiviral agents. J Biol Chem, 1999. 274(49): p. 34742-4.

61. De Clercq, E. and A. Holy, Acyclic nucleoside phosphonates: a key class of antiviral drugs. Nat Rev Drug Discov, 2005. 4(11): p. 928-40.

62. Aduma, P., et al., Metabolic diversity and antiviral activities of acyclic nucleoside phosphonates. Mol Pharmacol, 1995. 47(4): p. 816-22.

63. Gulnik, S.V. and M. Eissenstat, Approaches to the design of HIV protease inhibitors with improved resistance profiles. Curr Opin HIV AIDS, 2008. 3(6): p. 633-41.

64. Sheng, X.C., et al., Discovery of novel phosphonate derivatives as hepatitis C virus NS3 protease inhibitors. Bioorg Med Chem Lett, 2009. 19(13): p. 3453-3457.

47

65. Maruyama, H.B., M. Arisawa, and T. Sawada, Alafosfalin, a new inhibitor of cell wall biosynthesis: in vitro activity against urinary isolates in Japan and potentiation with beta-lactams. Antimicrob Agents Chemother, 1979. 16(4): p. 444-51.

66. Hahn, F.E., Alafosfalin, a new synthetic antibacterial compound. Naturwissenschaften, 1981. 68(2): p. 90.

67. Martin, M.B., et al., Bisphosphonates Inhibit the Growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum:  A Potential Route to Chemotherapy. J Med Chem, 2001. 44(6): p. 909-916.

68. Senaratne, S.G., et al., Bisphosphonates induce apoptosis in human breast cancer cell lines. Br J Cancer, 2000. 82(8): p. 1459-1468.

69. Guise, T.A., Antitumor effects of bisphosphonates: Promising preclinical evidence. Cancer treatment reviews, 2008. 34: p. S19-S24.

70. van der Pluijm, G., et al., Bisphosphonates inhibit the adhesion of breast cancer cells to bone matrices in vitro. The Journal of Clinical Investigation, 1996. 98(3): p. 698-705.

71. Palma, E., et al., Bisphosphonates as radionuclide carriers for imaging or systemic therapy. Molecular BioSystems, 2011. 7(11): p. 2950-2966.

72. Das, M., et al., Biofunctionalized, Phosphonate-Grafted, Ultrasmall Iron Oxide Nanoparticles for Combined Targeted Cancer Therapy and Multimodal Imaging. Small, 2009. 5(24): p. 2883-2893.

73. Gawalt, E.S., et al., Self-Assembly and Bonding of Alkanephosphonic Acids on the Native Oxide Surface of Titanium. Langmuir, 2001. 17(19): p. 5736-5738.

74. Viornery, C., et al., Osteoblast culture on polished titanium disks modified with phosphonic acids. Journal of Biomedical Materials Research, 2002. 62(1): p. 149-155.

75. Silverman, B.M., K.A. Wieghaus, and J. Schwartz, Comparative Properties of Siloxane vs Phosphonate Monolayers on A Key Titanium Alloy. Langmuir, 2004. 21(1): p. 225-228.

76. Adden, N., et al., Phosphonic Acid Monolayers for Binding of Bioactive Molecules to Titanium Surfaces. Langmuir, 2006. 22(19): p. 8197-8204.

77. Tsai, M.-Y. and J.-C. Lin, Surface characterization and platelet adhesion studies of self-assembled monolayer with phosphonate ester and phosphonic acid functionalities. Journal of Biomedical Materials Research, 2001. 55(4): p. 554-565.

78. Kem, K.M., Kinetics of the hydrolysis of linear poly[(acylimino)-ethylenes]. Journal of Polymer Science: Polymer Chemistry Edition, 1979. 17(7): p. 1977-1990.

79. Tanaka, R., et al., High molecular weight linear polyethylenimine and poly(N-methylethylenimine). Macromolecules, 1983. 16(6): p. 849-853.

80. Goff, J.D., et al., Novel Phosphonate-Functional Poly(ethylene oxide)-Magnetite Nanoparticles Form Stable Colloidal Dispersions in Phosphate-Buffered Saline. Chemistry of Materials, 2009. 21(20): p. 4784-4795.

81. Pothayee, N., et al., Synthesis of ‘ready-to-adsorb’ polymeric nanoshells for magnetic iron oxide nanoparticles via atom transfer radical polymerization. Polymer, 2011. 52(6): p. 1356-1366.

82. Kočovský, P., Addition Reactions: Polar Addition, in Organic Reaction Mechanisms · 20082011, John Wiley & Sons, Ltd. p. 267-329.

83. Carmichael, A.Y., et al., Synthesis and solution properties of poly (ethylene oxide-B-2-ethyl-2-oxazoline) and poly (ethylene oxide-B-ethyleneimine). POLYMER PREPRINTS-AMERICA-, 2004. 45(2): p. 476-477.

84. Gibson-Craig-Carmichael, A.Y., Synthesis of Amphiphilic Block Copolymers for Use in Biomedical Applications, 2010, Virginia Polytechnic Institute and State University.

85. Inagaki, N., K. Tomiha, and K. Katsuura, Studies on the thermal degradation of phosphorus containing polymers: 7. Thermal degradation of phosphorylated poly(vinyl alcohol). Polymer, 1974. 15(6): p. 335-338.

48

86. Troev, K.D., in Chemistry and Application of H-Phosphonates2006, Elsevier Science Ltd: Amsterdam.

87. Gentilhomme, A., et al., Thermal degradation of methyl methacrylate polymers functionalized by phosphorus-containing molecules-II: initial flame retardance and mechanistic studies. Polymer Degradation and Stability, 2003. 82(2): p. 347-355.